Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), has infected up to one-third of the world's population; the majority of these people reside in resource poor countries ravaged by HIV. TB is the leading cause of AIDS mortality in co-infected people. Rapid diagnosis and case identification are needed to control TB, minimize transmission, and improve AIDS survival. Over 90% of TB cases involve the lung and the diagnosis is made either by the microscopic examination of acid fast bacilli (AFB) strained sputum smears or culture. Cultures have a sensitivity of "70% but take several weeks for results. Smears have a lower sensitivity (approximately 45%) but can be done quickly. We have discovered a protein, CFP32, which is unique to the tuberculosis-causing organism, thereby has potential used as a diagnostic test. Based on a CFP32 "antigen capture assay", approximately 60% TB patients had the protein detected in their sputum while none of the patients with other lung diseases had CFP32 detected (100% specificity). Importantly, CFP32 was also detected in AFB smear and/or culture negative TB. Since M. tuberculosis is the only organism that produces CFP32, it was diagnostic of active pulmonary TB. Therefore, the "first generation" diagnostic assay developed using recombinant (r)CFP32 produced by E. coli was superior to sputum smear in sensitivity and approached the sensitivity of sputum culture. Although E. coli can produce large quantities of protein, these bacteria produce proteins that lack modifications introduced by tuberculosis-causing bacteria. The use of E. coli expressed CFP32 to produce antisera likely resulted in the "first generation" CFP32 capture assay having a lower sensitivity than its true potential. Therefore, this grant seeks to obtain rCFP32 expressed in genetic systems that closely approximate M. tuberculosis to immunize animals and to generate several high affinity antibodies in rabbit and mouse for a second generation CFP32 capture assay that can better detect the native protein in clinical samples. In addition, detection of other highly secreted mycobacteria protein, Ag85 complex proteins, will be evaluated as co-antigens using existing reagents. The predictive value of these "second generation" assays will be established using sputum samples from "suspected" TB patients (200 HIV-seronegative, 50 HIV-seropositive). The proposal will evaluate the sensitivity of the combined CFP32/Ag85 antigen capture assays, compare the sensitivity of AFB smear and culture in the same sample, and determine the ability of the new assay(s) to diagnosis TB in smear and culture negative cases.